In flowering plants, male fertility depends on proper cell differentiation in the anther. However, relatively little is known about the genes that regulate anther cell differentiation and function. Here, we report the analysis of a new Arabidopsis male sterile mutant, dysfunctional tapetum1 (dyt1). The dyt1 mutant exhibits abnormal anther morphology beginning at anther stage 4, with tapetal cells that have excess and/or enlarged vacuoles and lack the densely stained cytoplasm typical of normal tapetal cells. The mutant meiocytes are able to complete meiosis I,but they do not have a thick callose wall; they often fail to complete meiotic cytokinesis and eventually collapse. DYT1 encodes a putative bHLH transcription factor and is strongly expressed in the tapetum from late anther stage 5 to early stage 6, and at a lower level in meiocytes. In addition, the level of DYT1 mRNA is reduced in the sporocyteless/nozzle(spl/nzz) and excess microsporocytes1/extra sporogenous cell(ems1/exs) mutants; together with the mutant phenotypes, this suggests that DYT1 acts downstream of SPL/NZZ and EMS1/EXS. RT-PCR results showed that the expression levels of many tapetum-preferential genes are reduced significantly in the dyt1mutant, indicating that DYT1 is important for the expression of tapetum genes. Our results support the hypothesis that DYT1 is a crucial component of a genetic network that controls anther development and function.

In flowering plants, pollen grains are formed within the anther region of the male reproductive organ, the stamen(Ma, 2005). Molecular genetic studies have revealed that the B and C functions of the wellknown ABC model together determine the stamen identity, whereas the C function alone specifies the carpel identity (Coen and Meyerowitz,1991; Ma, 2005). In Arabidopsis, APETALA3 (AP3) and PISTILLATA(PI) are two essential B function genes, and AGAMOUS(AG) is required for C function(Coen and Meyerowitz, 1991; Ma, 2005). The Arabidopsis anther is a bilaterally symmetrical structure with four lobes. Each lobe comprises four distinct somatic cell layers from outer to inner: they are the epidermis, endothecium, middle layer and tapetum(Goldberg et al., 1993; Sanders et al., 1999). The tapetum surrounds meiocytes and generates many proteins, lipids,polysaccharides and other molecules necessary for pollen development(Goldberg et al., 1993). Accordingly, the tapetum is characterized by active protein synthesis and secretion, a high rate of energy metabolism, and expression of many tapetum-preferential genes (Dickinson and Bell, 1976; Hernould et al.,1998; Liu and Dickinson,1989; Rubinelli et al.,1998; Scott et al.,2004; Taylor et al.,1998; Zheng et al.,2003).

Several genes have been identified that are required for normal anther development (Ma, 2005). For example, the SPOROCYTELESS/NOZZLE (SPL/NZZ) gene is required for cell type specification in both male and female reproductive organs(Schiefthaler et al., 1999; Yang et al., 1999). The spl/nzz mutant anthers lack the endothecium, middle layer, tapetum and meiocytes. Recently, Ito et al. (Ito et al., 2004) found that SPL/NZZ is a direct target of AG, and that ectopic expression of SPL/NZZ, independent of AG,induces the formation of anther locule with pollen grains(Ito et al., 2004). SPL/NZZ encodes a putative transcription factor(Schiefthaler et al., 1999; Yang et al., 1999), suggesting that SPL/NZZ regulates anther cell differentiation by activating downstream genes.

Furthermore, EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS(EMS1/EXS) and TAPETUM DETERMINANT1 (TPD1) are early (pre-meiosis) genes that encode a receptor-like protein kinase and a small protein, respectively, and act in the same genetic pathway to control the tapetal cell identity (Canales et al.,2002; Sorensen et al.,2003; Yang et al.,2003; Yang et al.,2005; Zhao et al.,2002). Recently, the SERK1 and SERK2 genes encoding closely related receptor-like protein kinases were shown to have redundant functions in controlling tapetum formation(Albrecht et al., 2005; Colcombet et al., 2005). MALE STERILITY1 (MS1) and ABORTED MICROSPORES(AMS) act after meiosis and encode a PHD domain-containing protein and a bHLH transcription factor, respectively; they are essential for late stage functions of the tapetum (Ito and Shinozaki, 2002; Sorensen et al., 2003; Wilson et al.,2001). Remarkably, the early' gene EMS1/EXS is expressed in both stamen and gynoecium, whereas the late' genes MS1 and AMS are anther specific. However, it is not known how themale-specific' MS1 and AMS expression is regulated.

Here, we report the isolation of a new Arabidopsis mutant, which is male sterile and defective in tapetum differentiation and function. Because the mutant has an abnormal tapetum, we named the gene DYSFUNCTIONAL TAPETUM1 (DYT1). DYT1 encodes a putative bHLH transcription factor and is preferentially expressed in tapetal cells as early as anther stage 5, spatially similar to, but temporally earlier than, MS1 and AMS. Furthermore, our results suggest that DYT1 probably acts downstream of SPL/NZZ and EMS1/EXS, and is required for normal expression of AMS, MS1and other tapetumpreferential genes. We propose that DYT1 is a component of a genetic network for tapetum differentiation and function.

### Plant materials and growth

Arabidopsis thaliana plants in this study are in the Landsberg erecta (Ler) background for all experiments with the exception of the mapping of the DYT1 gene, for which we crossed dyt1 with Columbia. The spl(Yang et al., 1999), ems1 (Zhao et al.,2002) and tpd1 (Yang et al., 2003) mutants were kindly provided by Drs W. Yang, D. Zhao and D. Ye, respectively. The dyt1 mutant was isolated from the progeny of a Ds insertional line (ET4262). The ems1 dyt1double mutant was obtained by pollinating the dyt1 pistil with pollen from an ems1/+ plant. Plants were grown under long-day conditions (16 hours light/8 hours dark) in a ∼22°C growth room.

### Characterization of mutant phenotypes

Plants were photographed with a Sony digital camera, DSC-F707 (Sony Corp.,Tokyo, Japan). Flower pictures were taken using a Nikon dissecting microscope(Nikon Corp., Tokyo, Japan) with an Optronics digital camera (Optronics,Goleta, CA, USA). To determine pollen viability, mature anthers were stained with the Alexander solution (Alexander,1969) and photographed under an Olympus BX-51microscope (Olympus,Tokyo, Japan) with a SPOT II RT camera (Diagnostic Instruments, Sterling Heights, MI, USA). Chromosome spreading and DAPI staining were performed as described (Ross et al., 1996). Wild-type and mutant inflorescences were collected and fixed as described(Zhao et al., 2002). Floral buds were embedded in Spurr's resin; semi-thin (0.5 μm) sections were made by using an Ultracut E ultramicrotome (Leica Microsystems, Nussloch, Germany),stained with 0.05% of Toluidine Blue O for 40 to 60 seconds, and photographed under the Olympus BX-51 microscope with the SPOT II RT camera.

### Mapping and functional complementation

The dyt1 mutation was found to be distinct from the Dsinsertional locus (not shown). The dyt1 mutant (in the Lerbackground) was crossed with Columbia to obtain F1 and F2 seeds. About 500 F2 plants with mutant phenotypes were genotyped by using the SSLP and dCAPS markers (Li et al., 2001). Several predicted genes were found in the mapped region flanked by recombination events; these genes were amplified from wild-type and dyt1 plants and sequenced. TAIL PCR(Liu and Whittier, 1995) was used to determine the sequences of a retrotransposon insertion in the dyt1 mutant. To verify the DYT1-coding region, we used the primers oMC1872/oMC1873 (see Table 1 for all primer sequences), designed according to the annotated At4g21330 locus, to amplify wild-type Ler cDNA.

Table 1.

PCR primers in this study

oMC numberSequence (5′ to 3′)oMC numberSequence (5′ to 3′)
271 CGATGAGGAATTCAAAGCGC 2083 CATACTCTGTTTCGCTTCGTTGC
272 CGTCTCCTTCACATTATAACC 2084 GTACTCGTAATTGGCGTCTCATCC
490 CGTTCCGTTTTCGTTTTTTACC 2085 CCATTCCCATGGTCCAAACC
499 AACAAACCCCGTCAGCTTTA 2098 GTTCCAAGGGCACAGGTACATG
500 ACCGGAGAAGTGGTTGTCAC 2099 GGAATGTTCCGTTCGATGGAG
509 GTGGATCCGATCAGAACCTGTATTTCTT 2100 ATGGTATCTCTAAAGTCCCTT
510 CGCTCGAGTTTAAGGTTATATGGCTCAT 2101 CTAGGCACTTATGCCACAATC
1533 GGTAACATTGTGCTCAGTGGTGG 2102 GCGACTTTGGGAATCTGAGTTG
1534 AACGACCTTAATCTTCATGCTGC 2103 GGAATAGTTGAAGCGCAGCTCC
1823 ACTCTAACTGAAATCACATTCAC 2104 GCACTCCAGCATCCATAATGAATC
1834 CAGAGCCATAAGCCGACAATG 2105 ATAGGTCAGAAGCGGCAGGTAAC
1872 ATGGGTGGAGGAAGCAGATTTC 2127 CGGTCATCGGTAGCAACAGTAAC
1873 TTATGGATTGCTTCTCATAACTTC 2128 CCTCTGCCTTTACGGTTAAGACC
1945 GTCAAGAGTTCCTCTCAACATCAC 2129 TTCACGTATGACCCGCTCTTTC
1963 TAGACGGTTTTTCGCCCTTTGACG 2130 ATTGGTCTATTACATCGCTCTCCG
1964 ACGCGCAATAATGGTTTCTGA 2194 GTCGGAGCTAAGATTCTACGATA
2044 CGTTGAGAGGAATGGGTGTAGC 2238 TCGAGCTCTCTAGAATGGGTGGAGGAAGCAGATTTC
2045 AAACTGAAGCTGCTCTAGCGATTC 2239 TCGGCGCGCCAGATCTTTATGGATTGCTTCTCATAACTTC
2046 ATTTGCTCTACGGCATGGCTC 2241 CATCCGCTCGTCAAGGTATAC
2047 GGTAAGGGAAAGTGTTGGCGTAG 2242 CGTGGAGTAGGGAAAGTCAGAG
2048 TCGCTTGTTCCCAGGATAACC 2280 TCATCCCTCGTCTACGCTCC
2049 TTCCAGCAACGAGTTCCTTACG 2281 GTGGCCAGAGTAAGACTCGCA
2050 TCGTGCTCCACCTGGAGTTTAC 2362 TCCAACGGTGGTGGATTTTC
2051 TCCTTGCTTTCTCTTGCTCGATAG 2363 TGGTCCCGTATGATTTGTTGC
2052 GAGGATTACAGAGGTCGCTGAG 2364 CTCATCTCCTTCAAGCGCTCA
2053 GAGCCTTCTTTCCTTTCATCC 2365 AGACACGTAACGCCAACCCA
2054 ACTTAACCGGTCATCAGGTCATG 2366 GGTATCGGCGACACTGCTCT
2055 CGATATCCATCGGTATTCGACTC 2367 CCAATTCTCTCCGGCTCTACA
2056 GGCGACTCATCGACCTATCAAC 2368 TTCCCATCTTCCAGCTTCCA
2057 GACCCAAGAAGTCATAACCAATGC 2369 ACCACGTTCTGATCTTCAGCG
2058 ACAGAGGGATTGATCCAACGAG 2370 AGGCTGAAGACGTTCGGAGA
2059 TGCTGCTACCTCCGACTACATC 2371 GGCTTCGATGGGCTCTTCTA
2060 AAAGGTTCGGTCCCACTTGG 2372 GCTGCCGTTCTCTTTGAAGTCT
2061 ATGTTTCCGGTCACAGGGATG 2373 AAGAGCGGCTACAGCAGCTT
2062 ATGGCGTTTACTCCGAAGATC 2374 AACCGACGAACAACGTTCCT
2063 TCACACGGCAGTCGATATACTG 2375 CCCAGTACGAATTAAGCGCG
2064 GATATTCTCTCGGTGGTGCTGC 2376 CTTGCTGCTATTCTCGTTGCC
2065 TGGCCAGTAATAGGACCAACTAGC 2377 GGCTGCTCGAAGAGCGTTAC
2066 AGCTATAATAACCGGAGGAGCAAG 2378 TGTGAATAAGCATGGTGAGGGT
2067 GAGGAGAGATGCTATTCACACGG 2379 TGTGCAGCCATACGTGCC
2068 TCGTGATCCGAGCACTAGATTG 2380 TGATGACCTTCATTGGAGCG
2069 AGACTTCTTCTGTGCCCTACCAAC 2381 GCGCCAGTTCCTAGCATGTT
2070 CAAGAAAGGTCTCTGGACCGAG 2382 ATTTGCTCGAACGGATCCAG
2071 GAAGATGTGGTTCCATTGAGCAG 2383 CCTGACTCCCATCGAATGAAG
2072 CAGGAAGCAATGGCATAGTTCAC 2384 CAGCACTCGTGTTCTTGTTCCT
2073 TCCTTGCTCATGCTACTTGGAAG 2385 ACGCTGAGATTCACACCGCT
2074 AGTAGTGGTTCCAATGCCATTCAC 2463 GCGGAATCATCGAGTTTAGCA
2075 AAGCAATGCCTCAGAGCCTATG 2464 TGCGTAACTCGTTGATGTAATCG
2076 CATTAAAGCAGGCCACGTCAAG 2465 GATCACGGTCATCGGTAGCA
2077 ACCGAATCGAGCATCTGGTCTAG 2466 TGCGCATACGATGGATCTTG
2078 TGTGATAAGTACGGCCCACTTGTC 2467 CTTGTGGCAACTTCATGGGA
2079 TTAGTGTGAAACTCGTCAACACGC 2468 TTGAGCACCAGACAACAATGG
2080 TCCAAACGTCGCAACGGTC U1 ACCGGCAAGACCATCACTCT
2081 TCTCAGCTTCTGCTCCCACATG U2 AGGCCTCAACTGGTTGCTGT
2082 AAAGGCCGAGTCAACTCAGTTATG
oMC numberSequence (5′ to 3′)oMC numberSequence (5′ to 3′)
271 CGATGAGGAATTCAAAGCGC 2083 CATACTCTGTTTCGCTTCGTTGC
272 CGTCTCCTTCACATTATAACC 2084 GTACTCGTAATTGGCGTCTCATCC
490 CGTTCCGTTTTCGTTTTTTACC 2085 CCATTCCCATGGTCCAAACC
499 AACAAACCCCGTCAGCTTTA 2098 GTTCCAAGGGCACAGGTACATG
500 ACCGGAGAAGTGGTTGTCAC 2099 GGAATGTTCCGTTCGATGGAG
509 GTGGATCCGATCAGAACCTGTATTTCTT 2100 ATGGTATCTCTAAAGTCCCTT
510 CGCTCGAGTTTAAGGTTATATGGCTCAT 2101 CTAGGCACTTATGCCACAATC
1533 GGTAACATTGTGCTCAGTGGTGG 2102 GCGACTTTGGGAATCTGAGTTG
1534 AACGACCTTAATCTTCATGCTGC 2103 GGAATAGTTGAAGCGCAGCTCC
1823 ACTCTAACTGAAATCACATTCAC 2104 GCACTCCAGCATCCATAATGAATC
1834 CAGAGCCATAAGCCGACAATG 2105 ATAGGTCAGAAGCGGCAGGTAAC
1872 ATGGGTGGAGGAAGCAGATTTC 2127 CGGTCATCGGTAGCAACAGTAAC
1873 TTATGGATTGCTTCTCATAACTTC 2128 CCTCTGCCTTTACGGTTAAGACC
1945 GTCAAGAGTTCCTCTCAACATCAC 2129 TTCACGTATGACCCGCTCTTTC
1963 TAGACGGTTTTTCGCCCTTTGACG 2130 ATTGGTCTATTACATCGCTCTCCG
1964 ACGCGCAATAATGGTTTCTGA 2194 GTCGGAGCTAAGATTCTACGATA
2044 CGTTGAGAGGAATGGGTGTAGC 2238 TCGAGCTCTCTAGAATGGGTGGAGGAAGCAGATTTC
2045 AAACTGAAGCTGCTCTAGCGATTC 2239 TCGGCGCGCCAGATCTTTATGGATTGCTTCTCATAACTTC
2046 ATTTGCTCTACGGCATGGCTC 2241 CATCCGCTCGTCAAGGTATAC
2047 GGTAAGGGAAAGTGTTGGCGTAG 2242 CGTGGAGTAGGGAAAGTCAGAG
2048 TCGCTTGTTCCCAGGATAACC 2280 TCATCCCTCGTCTACGCTCC
2049 TTCCAGCAACGAGTTCCTTACG 2281 GTGGCCAGAGTAAGACTCGCA
2050 TCGTGCTCCACCTGGAGTTTAC 2362 TCCAACGGTGGTGGATTTTC
2051 TCCTTGCTTTCTCTTGCTCGATAG 2363 TGGTCCCGTATGATTTGTTGC
2052 GAGGATTACAGAGGTCGCTGAG 2364 CTCATCTCCTTCAAGCGCTCA
2053 GAGCCTTCTTTCCTTTCATCC 2365 AGACACGTAACGCCAACCCA
2054 ACTTAACCGGTCATCAGGTCATG 2366 GGTATCGGCGACACTGCTCT
2055 CGATATCCATCGGTATTCGACTC 2367 CCAATTCTCTCCGGCTCTACA
2056 GGCGACTCATCGACCTATCAAC 2368 TTCCCATCTTCCAGCTTCCA
2057 GACCCAAGAAGTCATAACCAATGC 2369 ACCACGTTCTGATCTTCAGCG
2058 ACAGAGGGATTGATCCAACGAG 2370 AGGCTGAAGACGTTCGGAGA
2059 TGCTGCTACCTCCGACTACATC 2371 GGCTTCGATGGGCTCTTCTA
2060 AAAGGTTCGGTCCCACTTGG 2372 GCTGCCGTTCTCTTTGAAGTCT
2061 ATGTTTCCGGTCACAGGGATG 2373 AAGAGCGGCTACAGCAGCTT
2062 ATGGCGTTTACTCCGAAGATC 2374 AACCGACGAACAACGTTCCT
2063 TCACACGGCAGTCGATATACTG 2375 CCCAGTACGAATTAAGCGCG
2064 GATATTCTCTCGGTGGTGCTGC 2376 CTTGCTGCTATTCTCGTTGCC
2065 TGGCCAGTAATAGGACCAACTAGC 2377 GGCTGCTCGAAGAGCGTTAC
2066 AGCTATAATAACCGGAGGAGCAAG 2378 TGTGAATAAGCATGGTGAGGGT
2067 GAGGAGAGATGCTATTCACACGG 2379 TGTGCAGCCATACGTGCC
2068 TCGTGATCCGAGCACTAGATTG 2380 TGATGACCTTCATTGGAGCG
2069 AGACTTCTTCTGTGCCCTACCAAC 2381 GCGCCAGTTCCTAGCATGTT
2070 CAAGAAAGGTCTCTGGACCGAG 2382 ATTTGCTCGAACGGATCCAG
2071 GAAGATGTGGTTCCATTGAGCAG 2383 CCTGACTCCCATCGAATGAAG
2072 CAGGAAGCAATGGCATAGTTCAC 2384 CAGCACTCGTGTTCTTGTTCCT
2073 TCCTTGCTCATGCTACTTGGAAG 2385 ACGCTGAGATTCACACCGCT
2074 AGTAGTGGTTCCAATGCCATTCAC 2463 GCGGAATCATCGAGTTTAGCA
2075 AAGCAATGCCTCAGAGCCTATG 2464 TGCGTAACTCGTTGATGTAATCG
2076 CATTAAAGCAGGCCACGTCAAG 2465 GATCACGGTCATCGGTAGCA
2077 ACCGAATCGAGCATCTGGTCTAG 2466 TGCGCATACGATGGATCTTG
2078 TGTGATAAGTACGGCCCACTTGTC 2467 CTTGTGGCAACTTCATGGGA
2079 TTAGTGTGAAACTCGTCAACACGC 2468 TTGAGCACCAGACAACAATGG
2080 TCCAAACGTCGCAACGGTC U1 ACCGGCAAGACCATCACTCT
2081 TCTCAGCTTCTGCTCCCACATG U2 AGGCCTCAACTGGTTGCTGT
2082 AAAGGCCGAGTCAACTCAGTTATG

The primers whose sequences have been shown in the text are not listed in this table. U1 and U2 are the RT-PCR primers for UBQ1.

To generate a complementation construct, a 2.7 kb wild-type genomic fragment was amplified by PCR using primers oMC2241 and oMC2242, and then cloned into a modified pCAMBIA1300, yielding pMC2969. The Agrobacterium strain GV1301 was transformed with the plasmid pMC2969,then used to transform dyt1/+ plants. The seeds of transformed plants were screened for hygromycin-resistant seedlings, which were transferred into the soil. The T1 plants were genotyped to identify homozygous dyt1plants with the T-DNA, using a primer (oMC1945) for the retrotransposon insertion at the DYT1 locus and DYT1-gene-specific primer oMC1834. The oMC1823/oMC1873 and oMC2194/oMC1834 primers were used to identify the dyt1 allele and the transgene, respectively.

### RT-PCR and real-time PCR

A set of genes were selected for expression analysis by RT-PCR because they are known to be important for male reproduction: SPL/NZZ(Schiefthaler et al., 1999; Yang et al., 1999), EMS1/EXS (Canales et al.,2002; Zhao et al.,2002), TPD1 (Yang et al., 2003; Yang et al.,2005), AMS (Sorensen et al., 2003), MS1(Ito and Shinozaki, 2002; Wilson et al., 2001), MALE STERILITY2 (Aarts et al.,1997), MALE STERILITY5(Glover et al., 1998), AtMYB32 (Preston et al.,2004), AtMYB103(Higginson et al., 2003), A6 and A9 (Sorensen et al.,2003), AtMYB33 and AtMYB65(Millar and Gubler, 2005), SOLO DANCERS (SDS) (Azumi et al., 2002), and ROCK-N-ROLLERS/AtMER3(RCK/AtMER3) (Chen et al.,2005; Mercier et al.,2005). A second group of 16 genes was identified using microarray data of wild-type and ems1 mutant anthers we had obtained in our laboratory (W.Z., Y.S., A. Wijeratne and H.M., unpublished). The genes that were expressed in the ems1 anther at levels that were one half or less of those in the wildtype anthers were regarded as candidate tapetum-preferential genes because the ems1 anthers lack tapetum. In addition, UBQ1 and AtMYB4(Vannini et al., 2004) were used as constitutive expression controls. The primers for RTPCR and relevant microarray data are provided in the Tables 1 and 2, respectively. The primers for real-time PCR are listed in Tables 1 and 3. The PCR and data treatment were carried out as described previously(Ni et al., 2004). Plant tissue was collected and quickly frozen in liquid nitrogen. The anthers at approximately anther stages 4 to 7 were collected under a dissection microscope. Total RNA was extracted using the RNeasy Plant Kit (Qiagen,Valencia, CA) from young inflorescences (approximately floral stage 1-10). 1-2μg total RNA was used for reverse transcription according to the manufacturer's instruction to synthesize cDNA, which was used directly as PCR templates (Invitrogen, Carlsbad, CA).

Table 2.

Microarray data of the genes selected in RT-PCR

Gene nameLocusoMCAv-WTAv-ems1Description
SPL At4g27330 2044/2045 402.50 283.52 SPOROCYTELESS/NOZZLE
EMS1 At5g07280 509/511 1062.95 146.89 EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS
TPD1 At4g24972 2046/2047   TAPETUM DETERMINANT 1
AMS At2g16910 2048/2049 348.88 9.39 ABORTED MICROSPORES
MS5 At4g20900 2050/2051 75.46 23.20 Male sterility MS5; pollenless3
MS5 like At5g44330 2052/2053 90.26 1.51 MS5 family protein
MS1 At5g22260 2054/2055   Male sterile protein 1
AtMYB32 At4g34990 2056/2057 300.93 63.67 MYB-like protein myb-related protein Y49
AtMYB4 At4g38620 2058/2059 161.19 179.94 Putative transcription factor (MYB4)
MS2 At3g11980 2060/2061 690.78 12.25 Male sterility protein 2 (MS2)
LPT12 At3g51590 2062/2063 446.85 40.33 Lipid transfer protein
ATSTP2 At1g07340 2064/2065 174.90 60.65 Hexose transporter
ATA1 At3g42960 2066/2067 2090.38 29.80 Alcohol dehydrogenase (ATA1)
PAB5 At1g71770 2068/2069 823.25 106.96 Polyadenylate-binding protein 5
At3g28470 2070/2071 758.75 22.98 Myb transcription factor
At3g13220 2072/2073 794.97 14.42 ABC transporter
At1g69500 2074/2075 1636.47 16.85 Cytochrome P450
At3g23770 2076/2077 497.53 17.17 β-1,3-glucanase
At1g01280 2078/2079 430.88 1.96 Cytochrome P450
FLF At5g10140 2080/2081 251.54 20.77 MADS box protein FLOWERING LOCUS F (FLF)
At3g60580 2082/2083 245.27 22.71 Zinc finger protein-like ZPT3-3, Petunia hybrida
ATMYB103 At5g56110 2084/2085 93.20 36.35 Regulate the tapetum and trichome development, anther specific gene
A6 At4g14080 2098/2099 3692.11 41.2 Glycosyl hydrolase family 17 protein / anther-specific protein (A6)
A9 At5g07230 2100/2101 5099.79 81.5 Protease inhibitor/seed storage/lipid transfer protein, tapetum-specific protein A9
MYB33 At5g06100 2102/2103 238.76 176.9 Myb family transcription factor (MYB33)
MYB65 At3g11440 2104/2105 335.48 203.4 Myb family transcription factor (MYB65)
At1G06170 2127/2128 938.35 28.1 Basic helix-loop-helix (bHLH) family protein
At2G31210 2129/2130 176.08 20.6 Basic helix-loop-helix (bHLH) family protein
SDS At1g14750 271/272 80.48 27.18 SOLO DANCER, a putative cyclin
RCK/AtMER3 At3g27730 1963/1964 81.68 37.16 ROCK-N-ROLLERS/AtMER3, a ATP-dependent DNA helicase
UBQ1 At3g52590 U1/U2 4509.45 4007.97 UBIQUITIN EXTENSION PROTEIN 1 (UBQ1)
Gene nameLocusoMCAv-WTAv-ems1Description
SPL At4g27330 2044/2045 402.50 283.52 SPOROCYTELESS/NOZZLE
EMS1 At5g07280 509/511 1062.95 146.89 EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS
TPD1 At4g24972 2046/2047   TAPETUM DETERMINANT 1
AMS At2g16910 2048/2049 348.88 9.39 ABORTED MICROSPORES
MS5 At4g20900 2050/2051 75.46 23.20 Male sterility MS5; pollenless3
MS5 like At5g44330 2052/2053 90.26 1.51 MS5 family protein
MS1 At5g22260 2054/2055   Male sterile protein 1
AtMYB32 At4g34990 2056/2057 300.93 63.67 MYB-like protein myb-related protein Y49
AtMYB4 At4g38620 2058/2059 161.19 179.94 Putative transcription factor (MYB4)
MS2 At3g11980 2060/2061 690.78 12.25 Male sterility protein 2 (MS2)
LPT12 At3g51590 2062/2063 446.85 40.33 Lipid transfer protein
ATSTP2 At1g07340 2064/2065 174.90 60.65 Hexose transporter
ATA1 At3g42960 2066/2067 2090.38 29.80 Alcohol dehydrogenase (ATA1)
PAB5 At1g71770 2068/2069 823.25 106.96 Polyadenylate-binding protein 5
At3g28470 2070/2071 758.75 22.98 Myb transcription factor
At3g13220 2072/2073 794.97 14.42 ABC transporter
At1g69500 2074/2075 1636.47 16.85 Cytochrome P450
At3g23770 2076/2077 497.53 17.17 β-1,3-glucanase
At1g01280 2078/2079 430.88 1.96 Cytochrome P450
FLF At5g10140 2080/2081 251.54 20.77 MADS box protein FLOWERING LOCUS F (FLF)
At3g60580 2082/2083 245.27 22.71 Zinc finger protein-like ZPT3-3, Petunia hybrida
ATMYB103 At5g56110 2084/2085 93.20 36.35 Regulate the tapetum and trichome development, anther specific gene
A6 At4g14080 2098/2099 3692.11 41.2 Glycosyl hydrolase family 17 protein / anther-specific protein (A6)
A9 At5g07230 2100/2101 5099.79 81.5 Protease inhibitor/seed storage/lipid transfer protein, tapetum-specific protein A9
MYB33 At5g06100 2102/2103 238.76 176.9 Myb family transcription factor (MYB33)
MYB65 At3g11440 2104/2105 335.48 203.4 Myb family transcription factor (MYB65)
At1G06170 2127/2128 938.35 28.1 Basic helix-loop-helix (bHLH) family protein
At2G31210 2129/2130 176.08 20.6 Basic helix-loop-helix (bHLH) family protein
SDS At1g14750 271/272 80.48 27.18 SOLO DANCER, a putative cyclin
RCK/AtMER3 At3g27730 1963/1964 81.68 37.16 ROCK-N-ROLLERS/AtMER3, a ATP-dependent DNA helicase
UBQ1 At3g52590 U1/U2 4509.45 4007.97 UBIQUITIN EXTENSION PROTEIN 1 (UBQ1)

The genes were selected as described.

The microarray intensity value of the gene for the ems1/exs anther is at most half of that from the wild-type anther. Genes known to be important for early anther development are regulatory genes SPL/NZZ, TPD1,AtMYB33 and AtMYB65. Constitutive controls are AtMYB4and the UBQ1.

Table 3.

Primers used in real-time PCR

Gene nameLocusoMC numberProduct length
SPL At4g27330 2362/2363 200
EMS1 At5g07280 2364/2365 101
TPD1 At4g24972 2366/2367 201
AMS At2g16910 2368/2369 201
MS1 At5g22260 2370/2371 201
MYB33 At5g06100 2378/2379 201
MYB65 At3g11440 2380/2381 201
bHLH At1g10610 2384/2385 201
DYT1 At4g21330 1872/1834 153
bHLH At2g31210 2463/2464 201
bHLH At1g06170 2465/2466 201
WD-40 At1g15850 2467/2468 201
ACT2 AT3G18780 1533/1534 108
Gene nameLocusoMC numberProduct length
SPL At4g27330 2362/2363 200
EMS1 At5g07280 2364/2365 101
TPD1 At4g24972 2366/2367 201
AMS At2g16910 2368/2369 201
MS1 At5g22260 2370/2371 201
MYB33 At5g06100 2378/2379 201
MYB65 At3g11440 2380/2381 201
bHLH At1g10610 2384/2385 201
DYT1 At4g21330 1872/1834 153
bHLH At2g31210 2463/2464 201
bHLH At1g06170 2465/2466 201
WD-40 At1g15850 2467/2468 201
ACT2 AT3G18780 1533/1534 108

### RNA in situ hybridization

Non-radioactive RNA in situ hybridization was performed as described(Li et al., 2004). A 624 bp DYT1 cDNA fragment was amplified using DYT1-specific primers with XbaI and BglII sites at the 5′ end: oMC2238 and oMC2239, respectively. The PCR product was confirmed by sequencing and cloned into the pGEM-T vector (Promega, Madison, WI) to yield plasmid pMC2949. Plasmid DNA was completely digested by XbaI or BglII and used as template for transcription with SP6 or T7 RNA polymerases,respectively (Roche, Mannheim, Germany). Images were obtained using the Olympus BX-51 microscope with the SPOT II RT camera and edited using PHOTOSHOP 5.0 (Adobe, San Jose, CA).

### Overexpression of DYT1

A DYT1 cDNA fragment was amplified using gene-specific primers oMC1872 and oMC1873 (Table 1),then cloned into pGEM-T vector to yield plasmid pMC2941. After verification by sequencing, the DYT1 cDNA fragment was subcloned into pCAMBIA1300 downstream of the CaMV 35S promoter to produce the plasmid pMC2942. An Agrobacterium strain C3581 carrying pMC2942 was used to transform wild-type, ems1/+ and spl/+ plants. The transgenic plants were selected by hygromycin resistance and verified using PCR with oMC1872 and oMC1873. EMS1 gene-specific primers oMC499 and oMC500, SPL/NZZ gene-specific primers oMC2044 and oMC2045, plus Ds5-specific primer oMC490 were used to identify ems1 and splheterozygous and homozygous plants, respectively. Paraffin sections were prepared as described for in situ experiments (above) and photographed as described for semi-thin sections.

### Phylogenetic analysis of the DYT1 subfamily

The protein sequences of the nine genes from group 9 of the Arabidopsis bHLH family, including DYT1, were used to search for the closest homologs in both the rice genome(http://tigrblast.tigr.org/euk-blast/index.cgi?project=osa1)and Populus genome(http://www.floralgenome.org/cgi-bin/tribedb/tribe.cgi)using both BLAST and TBLASTN programs with a cutoff of 1E-15(Heim et al., 2003; Toledo-Ortiz et al., 2003). The multiple sequence alignment of full-length protein sequences was performed using ClustalX (Plate-Forme de Bio-Informatique, Illkirch Cedex, France) with a combination of GOP=4.0 and GEP=0.1. The bHLH domain region and additional conserved regions were aligned and used to perform neighbor joining (NJ)analyses with the pairwise deletion' option, P-distance' model and 1000 bootstrap replicates test using MEGA version 3.0(Kumar et al., 2004)(http://www.megasoftware.net/index.html).

### The isolation of a new male-sterile mutant

To identify new Arabidopsis genes important for anther development, we screened for male sterile plants among Ds transposon insertional lines. One line was found to segregate normal and sterile plants with an approximate 3:1 ratio. Pollination of the mutant pistil with wild-type pollen indicated that the mutant is female fertile. The mutant was named dysfunctional tapetum1 (dyt1) because of its anther defects(see below). The vegetative growth of the dyt1 mutant appeared normal(Fig. 1A,B) and most mutant floral organs were also normal with the exception of shorter stamen filaments and smaller anthers (Fig. 1C,D). There were no pollen grains on the anther surface of opened flowers (Fig. 1G). Occasionally mutant siliques contained a few seeds (not shown), possibly owing to residual gene function (see below).

### The dyt1 mutant is defective in tapetum development

Detailed analyses were performed to understand the mutant developmental defects. Chromosome spread experiments were performed and revealed that normal meiotic features could be observed in the mutant meiocytes, demonstrating that meiotic nuclear divisions can proceed normally(Fig. 2). We also generated semi-thin anther sections to investigate the mutant anther development(Fig. 3). From anther stage 1 to 3, the dyt1 anthers appeared normal (data not shown). At stage 4,the mutant anther was similar to the wild-type anther in cell layer and cell number, but had a slightly different shape from the wild-type anther and was vacuolated in more cells than normal. In addition, the mutant sporogenous cells appeared more deeply stained than wild-type cells(Fig. 3A,B). At early stage 5,the dyt1 anther lobe had four cell types interior to the epidermis,similar to the wild type. The wild-type anther at late stage 5(Fig. 3E) was also vacuolated in more cells than earlier and had deeply stained meiocytes. Therefore, mutant anthers at stage 4 and early stage 5 exhibited these morphological features precociously (Fig. 3A-D).

In the wild-type anther at late stage 5(Fig. 3E), the tapetum had significantly larger cells than earlier. In the mutant(Fig. 3F), additional vacuoles were observed in tapetal cells, with a reduction of the cytoplasm. The vacuoles in the wild-type tapetum at this time are fewer and smaller than those in the mutant. In addition, the mutant middle layer maintained its thickness with vacuolation, unlike the reduced thickness of the wild-type middle layer (Fig. 3E,F). At stage 6, a thick callose wall forms around the meiocytes(Fig. 3G). By contrast, the mutant meiocytes had very thin callose cell walls(Fig. 3G,H). At this stage,most of the mutant meiocytes were undergoing meiosis(Fig. 2), but some of them had collapsed. At stage 7 and stage 8, the completion of wild-type meiosis results in the formation of tetrads and then microspores, but mutant tapetal and middle layer cells swelled with expanded vacuoles and filled the center of the locules where the meiocytes had collapsed and degraded(Fig. 3I-L).

### DYT1 encodes a putative bHLH transcription factor

To gain further insights into its function, we cloned the DYT1gene. The dyt1 mutant was from a Ds insertional line, but the dyt1 mutation was genetically separable from the Dselement (data not shown). To clone the gene, we mapped the dyt1 locus by analyzing ∼500 mutant F2 progenies from a cross between the dyt1 mutant (Ler) and Columbia wild-type plant. The mapping results indicated that the DYT1 gene was on chromosome 4, between At4g21220 and At4g21360 (data not shown). We sequenced candidate genes in this region from both wild-type and the dyt1 mutant, and found that only one locus, At4g21330, had an insertion mutation 109 bp upstream of the predicted translation initiation codon(Fig. 4A). We performed TAIL PCR to obtain the DNA at the 5′ and 3′ ends of the insertion and found that they each partially matched the same region of a putative retro-transposon At5g33382 in the Columbia genomic sequence. Further PCR and sequence analysis indicated that the insertion at the DYT1 locus matched exactly and completely to a seemingly intact retro-transposon in the Ler genome (W.Z., Y.S. and H.M., unpublished). To verify that At4g21330 is DYT1, we cloned an At4g21330 genomic fragment into a modified pCAMBIA1300 plasmid and used it to transform dyt1/+ plants. Fifty independent transgenic plants were analyzed; 12 lines were found to be homozygous for the dyt1 insertion. All 12 lines were fertile,including nine lines with normal fertility(Fig. 1E,H), confirming that At4g21330 is the DYT1 gene.

Fig. 1.

Phenotypes of the wild type (Ler), dyt1 mutant and transgenic plants for complementation. (A) A Ler plant.(B) A dyt1 plant, with very small siliques (arrows).(C) A Ler flower. (D) A dyt1 flower.(E) A flower of the dyt1 plant with the DYT1transgene. (F) A wild-type anther, with viable pollen grains (stained).(G) A dyt1 anther, no viable pollen. (H) An anther from a dyt1 plant with the DYT1 transgene, with a large number of viable pollen grains and some microspores (arrowheads). Scale bars: 10 mm in A,B; 500 μm in C-E; 20 μm in F-H.

Fig. 1.

Phenotypes of the wild type (Ler), dyt1 mutant and transgenic plants for complementation. (A) A Ler plant.(B) A dyt1 plant, with very small siliques (arrows).(C) A Ler flower. (D) A dyt1 flower.(E) A flower of the dyt1 plant with the DYT1transgene. (F) A wild-type anther, with viable pollen grains (stained).(G) A dyt1 anther, no viable pollen. (H) An anther from a dyt1 plant with the DYT1 transgene, with a large number of viable pollen grains and some microspores (arrowheads). Scale bars: 10 mm in A,B; 500 μm in C-E; 20 μm in F-H.

To determine the DYT1 cDNA sequence, we performed an RTPCR experiment with floral mRNA, and obtained a 624 bp product with an identical sequence to that indicated by the annotation. Additional RT-PCR experiments using primers that match sequences just beyond the annotated region yielded no product, confirming the annotated DYT1-coding region. The DYT1 gene encodes a putative transcription factor of 207 amino acid residues with a basic helix-loop-helix (bHLH) domain. According to the annotation(http://www.arabidopsis.org),the bHLH domain spans the region from Phe29 to Gln78(Fig. 4B)(Toledo-Ortiz et al., 2003). Interestingly, a preliminary search of public databases using the DYT1 protein sequence with the BLAST program showed that the DYT1 protein has the highest similarity to the Arabidopsis AMS protein(Sorensen et al., 2003).

To gain additional insights into the phylogenetic relationship between DYT1, AMS and other close homologs, we performed phylogenetic analysis of bHLH genes, including DYT1, AMS and a recently reported rice gene, UNDEVELOPED TAPETUM (OsUDT1), which is required for normal tapetum development (Jung et al., 2005). The phylogenetic analyses with the bHLH domain region alone, with both bHLH domain and conserved regions(Fig. 4C), or with the full-length sequences yielded trees with very similar topologies (others not shown). Our result indicates that DYT1, AMS, OsUDT1, Os02g02820 and PtDYT1-Like (Populus trichocarpa) form a separate clade within a group of related members of the bHLH gene family. Among them, AMS and Os02g02820 are supported as an orthologous pair, as are DYT1 and PtDYT1-like. In Arabidopsis, DYT1 and AMS are most closely related to each other, in agreement with the preliminary BLAST results. The rice OsUDT1 gene could be the ortholog of the DYT1 and PtDYT1-like genes.

Fig. 2.

Meiosis in Ler and dyt1 anthers.(A,B) Pachytene images of the Ler (A) and dyt1(B) meiocytes with condensed chromosomes. (C,D) Diakinesis images of the Ler (C) and dyt1 (D) meiocytes, each with five bivalents of attached homologous chromosomes. (E,F) Telophase II images of the Ler (E) and dyt1 (F) meiocytes. Both show four decondensed chromosome clusters. Scale bar: 5 μm.

Fig. 2.

Meiosis in Ler and dyt1 anthers.(A,B) Pachytene images of the Ler (A) and dyt1(B) meiocytes with condensed chromosomes. (C,D) Diakinesis images of the Ler (C) and dyt1 (D) meiocytes, each with five bivalents of attached homologous chromosomes. (E,F) Telophase II images of the Ler (E) and dyt1 (F) meiocytes. Both show four decondensed chromosome clusters. Scale bar: 5 μm.

### DYT1 is preferentially expressed in tapetal cells

To determine the DYT1 expression pattern, we performed a realtime PCR experiment (Fig. 5A). DYT1 expression was detected at a low level in young inflorescences with meiotic cells and in siliques, and at a high level in the wild-type anthers from stage 4 to 7, but not in vegetative tissues or in the post-meiotic stage-12 flower. In addition, RNA in situ hybridization experiments showed that DYT1 expression was detectable in the floral meristem and early anther primordia (Fig. 5C), and in archesporial cells at stage 2 (not shown). From stage 4 to early stage 5, a weak signal was detected in precursors of the middle layer, tapetum and meiocytes (Fig. 5D). From late stage 5 (Fig. 5E,H) to early stage 6, the DYT1 expression reached its highest level in the tapetum and is at a low level in meiocytes. However, at late stage 6, the DYT1 expression signal was drastically reduced to background levels (Fig. 5F). The DYT1 expression pattern is consistent to the observed tapetal defects in the dyt1 mutant anther. In the gynoecium, a weak signal was detected (data not shown). In the dyt1 anther, there was a low level expression, which was not specific to the tapetum(Fig. 5I). Therefore, the insertion upstream of the DYT1 ATG codon caused a great reduction of the level of DYT1 expression in the tapetum. The weak expression suggests that the dyt1 allele may have some residual function, which might account for the occasional seeds that were observed.

Fig. 3.

Anther development from stage 4 to stage 8 in the wild type(Ler) and dyt1 mutant. Locules from anther sections:(A,C,E,G,I,K) wild type; (B,D,F,H,J,L) dyt1 mutants.(A,B) Stage 4 anthers. (C,D) Late stage 4 or very early stage 5. Vacuolization in the dyt1 mutant occurred in more cells and the vacuoles were larger than those in the wild type.(E,F) Stage 5 anthers, with more and larger vacuoles in the tapetum and middle layer of the dyt1 anther (F) than the wild type(E). (G,H) Stage 6 anthers. The vacuolization in cells of the mutant tapetum and middle layer became more extensive. The mutant meiocytes had a much thinner callose layer around them (arrowheads). (I,J)Stage 7 anthers. Wild-type meiocytes undergo cytokinesis to form tetrads. In dyt1 anther, the tapetum and middle layer cells were swollen and had excess vacuolization, and meiocytes generally collapsed before cytokinesis(arrowheads). (K,L) At stage 8, in the wild-type anther locules,microspores were released from the tetrad; in the dyt1 anther, almost all meiocytes degenerated. E, epidermis; En, endothecium; ISP, inner secondary parietal cells; SS, secondary sporogenous cells; ML, middle layer; T, tapetum;Ms, meiocytes; Tds, tetrads; Msp, microspores; D-Ms, degenerated meiocytes. Scale bars: 10 μm.

Fig. 3.

Anther development from stage 4 to stage 8 in the wild type(Ler) and dyt1 mutant. Locules from anther sections:(A,C,E,G,I,K) wild type; (B,D,F,H,J,L) dyt1 mutants.(A,B) Stage 4 anthers. (C,D) Late stage 4 or very early stage 5. Vacuolization in the dyt1 mutant occurred in more cells and the vacuoles were larger than those in the wild type.(E,F) Stage 5 anthers, with more and larger vacuoles in the tapetum and middle layer of the dyt1 anther (F) than the wild type(E). (G,H) Stage 6 anthers. The vacuolization in cells of the mutant tapetum and middle layer became more extensive. The mutant meiocytes had a much thinner callose layer around them (arrowheads). (I,J)Stage 7 anthers. Wild-type meiocytes undergo cytokinesis to form tetrads. In dyt1 anther, the tapetum and middle layer cells were swollen and had excess vacuolization, and meiocytes generally collapsed before cytokinesis(arrowheads). (K,L) At stage 8, in the wild-type anther locules,microspores were released from the tetrad; in the dyt1 anther, almost all meiocytes degenerated. E, epidermis; En, endothecium; ISP, inner secondary parietal cells; SS, secondary sporogenous cells; ML, middle layer; T, tapetum;Ms, meiocytes; Tds, tetrads; Msp, microspores; D-Ms, degenerated meiocytes. Scale bars: 10 μm.

### DYT1 expression is positively regulated by SPL/NZZand EMS1/EXS

To test whether the DYT1 expression was affected by any known early anther development genes, we performed real-time PCR experiments with RNA from spl and ems1 mutant floral buds. Our results showed that the DYT1 expression was barely detectable in the splinflorescences and that the expression level in the ems1inflorescences was only ∼17% of the wild-type level(Fig. 5B). These results suggest that DYT1 might be downstream of SPL/NZZ and EMS1/EXS. We performed RNA in situ experiments to verify this possibility. In the spl anther, little DYT1 expression could be detected (Fig. 5J). A weak signal could be detected in the meiocytes in the ems1 anther at late stage 5 (Fig. 5K) and early stage 6, but, unlike the wild-type anther, the strong tapetal signal was not found in the ems1 anther (compare Fig. 5H with 5K). Therefore,the strong DYT1 expression in the tapetum requires EMS1/EXS. Both the real-time PCR and the RNA in situ hybridization results indicate that SPL/NZZ is essential for DYT1 expression and that EMS1/EXS promotes the high-level DYT1 expression specific to the tapetum.

### The expression of anther genes in the dyt1 mutant is altered

The finding that DYT1 encodes a bHLH-type putative transcription factor suggests that DYT1 controls gene expression required for normal anther development. To test this hypothesis, we performed RT-PCR using primers for anther genes. We obtained results for a total of 32 genes(Fig. 6); among these, SPL/NZZ, EMS1/EXS and TPD1 are known early anther development genes (Canales et al.,2002; Schiefthaler et al.,1999; Yang et al.,1999; Yang et al.,2003; Yang et al.,2005; Zhao et al.,2002). The other genes were chosen for their tapetum-preferential expression according to either previous reports or to gene expression data obtained in our laboratory (Table 2). We found that for 21 genes out of 32, the expression was significantly reduced in the dyt1 mutant compared with the wild type(Fig. 6), indicating that indeed the normal expression of a large number of genes depends on the DYT1 gene function. In particular, two regulatory genes, MS1and AMS, which are important for tapetum development(Ito and Shinozaki, 2002; Sorensen et al., 2003; Wilson et al., 2001),exhibited greatly reduced levels of expression, suggesting that MS1and AMS act downstream of DYT1. By contrast, some tapetum preferential genes, such as A6 and A9, were still expressed in the dyt1 mutant at slightly reduced levels. In addition, the expression of SPL, TPD1, EMS1/EXS, AtMYB33 and AtMYB65 was not dramatically different in the dyt1 mutant, indicating that their expression does not require DYT1. To verify the RT-PCR results,selected genes were further analyzed using real-time PCR and the results(Fig. 6D) support the conclusion that AMS and MS1 expression requires DYT1 function.

In addition to the tapetum-preferential genes, we also tested the expression of two meiosis-specific genes: SDS and RCK/AtMER3(Azumi et al., 2002; Chen et al., 2005; Mercier et al., 2005). Although the expression level of SDS did not change, the expression of RCK/AtMER3 was significantly reduced. SDS is known to act earlier than RCK/AtMER3 in prophase I, suggesting that the dyt1 mutation might affect the expression of late prophase I genes more than early prophase I genes.

### DYT1 is not sufficient for tapetum development

Both ems1/exs and dyt1 mutations affect tapetum development. Previous reports and our results suggest that DYT1 acts downstream of EMS1/EXS. To test this further, we generated the ems1 dyt1 double mutant and examined its early anther development. We found that the double mutant resembles the ems1 mutant in that the double mutant anther also completely lacks the tapetum(Fig. 7), suggesting that indeed EMS1/EXS is upstream of DYT1 in the same pathway. In addition, to test whether DYT1 is sufficient to alter anther development, we generated transgenic plants carrying a 35S-DYT1fusion in wild-type, spl/nzz and ems1/exsbackgrounds. Transgenic lines with DYT1 overexpression were identified by RTPCR (Fig. 6E;data not shown) and analyzed for their anther morphology. In all cases, the 35S-DYT1 transgenic anthers had morphologies resembling those of the corresponding genotypes without the transgene (see Fig. S1 in the supplementary material). Therefore, the overexpression of DYT1 was not able to suppress the spl/nzz and ems1/exs mutant phenotypes, indicating that other genes acting downstream of SPL/NZZand EMS1/EXS are probably required for normal tapetum development. Because DYT1 was found to be required for normal expression of a number of tapetum genes, we tested selected genes by real-time PCR to determine whether the 35S-DYT1 transgene was able to stimulate the expression of these genes. Our results indicate that the 35S-DYT1transgene did not alter the expression of these genes substantially(Fig. 6E), consistent with the morphological results.

Fig. 4.

The DYT1 gene structure and annotated conserved domain.(A) The genomic region of the DYT1 gene. The dyt1insertion is flanked by a direct repeat of 6 bp ACTTCT, which correspond to nucleotides 109-104 upstream of the annotated ATG (nucleotides 1-3) codon. The DYT1 gene has three exons represented as white boxes. (B) The annotated amino acid sequence of the DYT1 protein, with 207 amino acid residues. From Phe29 to the Gln78 is the conserved bHLH domain, which corresponds to the black region in the schematic box image and is underlined in the amino acid sequence. (C) An unrooted neighbor-joining tree of Arabidopsis, rice, and Poplar bHLH genes in the same subfamily as DYT1. Gene ID numbers starting withAt' indicate genes from Arabidopsis thaliana; names of genes with functional information are given after the gene ID numbers. Gene ID numbers starting with Os' indicates genes from rice (Oryza sativa); UDT1 is shown as OsUDT1. Pt' indicates genes from poplar (Populus trichocarpa), with temporary names given according to gene ID from the Floral Genome Project. Bootstrap values are shown near the relevant nodes.

Fig. 4.

The DYT1 gene structure and annotated conserved domain.(A) The genomic region of the DYT1 gene. The dyt1insertion is flanked by a direct repeat of 6 bp ACTTCT, which correspond to nucleotides 109-104 upstream of the annotated ATG (nucleotides 1-3) codon. The DYT1 gene has three exons represented as white boxes. (B) The annotated amino acid sequence of the DYT1 protein, with 207 amino acid residues. From Phe29 to the Gln78 is the conserved bHLH domain, which corresponds to the black region in the schematic box image and is underlined in the amino acid sequence. (C) An unrooted neighbor-joining tree of Arabidopsis, rice, and Poplar bHLH genes in the same subfamily as DYT1. Gene ID numbers starting withAt' indicate genes from Arabidopsis thaliana; names of genes with functional information are given after the gene ID numbers. Gene ID numbers starting with Os' indicates genes from rice (Oryza sativa); UDT1 is shown as OsUDT1. `Pt' indicates genes from poplar (Populus trichocarpa), with temporary names given according to gene ID from the Floral Genome Project. Bootstrap values are shown near the relevant nodes.

Our results and previous studies suggest that additional genes other than DYT1 probably function downstream of SPL/NZZ and EMS1/EXS to promote tapetum development. It is possible that some of these genes might encode regulatory proteins. For example, the AtMYB33 and AtMYB65 genes are known to be important for tapetum development, and might also function downstream of SPL/NZZand EMS1/EXS. To test these ideas, we examined the expression in wild-type and mutants of these genes and of other genes, which were identified as tapetum-preferential from our microarray data (W.Z., Y.S. and H.M.,unpublished) and encode bHLH or WD-40 proteins. Real-time PCR results(Fig. 6F) indicate that the expression of two bHLH genes (At2g31210 and At1g06170) was greatly reduced in the spl mutant, and one of them (At2g31210) was expressed at a lower level in the ems1 mutant. By contrast, the expression levels of AtMYB33, AtMYB65 and two other genes were either nearly normal or increased.

### DYT1 is important for tapetum development and function

Our analysis of the dyt1 mutant phenotype indicates that DYT1 is required for normal tapetum development following its formation. Furthermore, DYT1 is expressed preferentially in the tapetum at late stage 5, consistent with its role in this layer. The EMS1/EXS, SERK1/SERK2 and TPD1 genes(Albrecht et al., 2005; Canales et al., 2002; Colcombet et al., 2005; Yang et al., 2003; Yang et al., 2005; Zhao et al., 2002) are important for the formation of the tapetal layer; therefore, these genes probably act at earlier stages than DYT1, as supported by the observed strong tapetal expression of EMS1/EXS and TPD1(Canales et al., 2002; Yang et al., 2003; Zhao et al., 2002) prior to the strong DYT1 expression in the tapetum. Our expression and double mutant analyses support the hypothesis that DYT1 acts downstream of SPL/NZZ and EMS1/EXS.

AMS and MS1 are also important for tapetum function, but they are required at post-meiotic steps(Ito and Shinozaki, 2002; Sorensen et al., 2003; Wilson et al., 2001). In ams anthers, meiosis is normal and microspores are formed; however,the newly formed microspores soon degenerate. In ms1 anthers, pollen development is abnormal and no normal mature pollen is produced. Compared with AMS and MS1, DYT1 acts at an earlier stage, before the completion of meiosis. Therefore, DYT1 is required for a key step in tapetum development. In other words, tapetum development requires the combined activities of the EMS1/EXS, SERK1/SERK2, TPD1, DYT1, AMS and MS1 genes: first EMS1/EXS, SERK1/SERK2 and TPD1 specify the tapetal cells as distinct from meiocytes at the time of the cell division that form the tapetal cells, then DYT1 is required to promote correct tapetal cell fate for proper function, and finally AMS and MS1 further regulate the tapetal cell function supporting normal microspore development.

### DYT1 is required for normal levels of the expression of tapetum genes

As DYT1 encodes a bHLH putative transcription factor, it is likely that it regulates the expression of tapetal genes. We found that the expression of a majority of tapetum-preferential genes tested depends on DYT1. The greatly reduced expression in the dyt1 mutant of many tapetum-preferential genes, particularly those encoding transcription factors, supports the idea that DYT1 is a key component of a regulatory step in normal tapetum development. The Arabidopsis bHLH gene family has over 140 members, making this the second largest gene family of transcription factors (Toledo-Ortiz et al., 2003). Although DYT1 and AMS clearly have non-redundant and distinct functions, they are members of the same subfamily. Phylogenetic analysis performed here including the closest rice homologs of DYT1 indicates that the rice gene, OsUDT1, is also a member of this subfamily and a putative DYT1 ortholog. A mutation in the OsUDT1 gene results in a defective tapetum(Jung et al., 2005), similar to the dyt1 tapetum. In addition, the expression pattern of the OsUDT1 gene (Jung et al.,2005) is different from that of DYT1. Therefore, this bHLH subfamily contains phylogenetically and functionally distinct members.

Fig. 5.

The DYT1 expression pattern. (A) Detection of DYT1 expression using real-time PCR in Ler background. DYT1 expression was not detected in any vegetative tissues or stage-12 flower, was detected at low levels in the young inflorescence and siliques, and was at the highest level in the anther. (B) Detection of DYT1 expression using real-time PCR in Ler, ems1 and spl inflorescences. The DYT1 expression was not detected in spl, but was detected in ems1 at about 17% of the normal level. (C-K) RNA in situ hybridization with a DYT1 probe.(C-F,H) DYT1 expression in the Ler background. (C) The DYT1 signal was detected in the floral meristem. (D) An anther at stage 4 to early stage 5. The DYT1 signal can be detected mainly within the newly formed tapetum and meiocytes. (E,H) At late stage 5, a strong signal is detected in the tapetal cell layer, whereas the signal in the meiocytes is much weaker. (F) At late stage 6, the DYT1 signal is greatly reduced, with residual expression in some meiocytes. (I) A dyt1 mutant anther at late stage 5; the DYT1 signal is low and non-specific in the entire anther. (J) A spl mutant anther at late stage 5. The DYT1 signal is at the background level. (K) An ems1 mutant anther locule at late stage 5. Uniformly weak DYT1 signal can be detected in meiocytes and little signal in cells surrounding the meiocytes. (G) The sense control with a Ler late stage 5 anther. Only background signal is seen. Rt, root; Sm, stem; Lf, leaf;Se, silique; S12, stage 12 flower; Inf, inflorescence; Ar, anther; WT-Inf,wild-type inflorescence; ems1-Inf, ems1 inflorescence; spl-Inf, spl inflorescence; T, tapetum; Ms, meiocytes; I,indeterminate cells; E-Ms, excess meiocytes. Scale bars: 20 μm in C-K.

Fig. 5.

The DYT1 expression pattern. (A) Detection of DYT1 expression using real-time PCR in Ler background. DYT1 expression was not detected in any vegetative tissues or stage-12 flower, was detected at low levels in the young inflorescence and siliques, and was at the highest level in the anther. (B) Detection of DYT1 expression using real-time PCR in Ler, ems1 and spl inflorescences. The DYT1 expression was not detected in spl, but was detected in ems1 at about 17% of the normal level. (C-K) RNA in situ hybridization with a DYT1 probe.(C-F,H) DYT1 expression in the Ler background. (C) The DYT1 signal was detected in the floral meristem. (D) An anther at stage 4 to early stage 5. The DYT1 signal can be detected mainly within the newly formed tapetum and meiocytes. (E,H) At late stage 5, a strong signal is detected in the tapetal cell layer, whereas the signal in the meiocytes is much weaker. (F) At late stage 6, the DYT1 signal is greatly reduced, with residual expression in some meiocytes. (I) A dyt1 mutant anther at late stage 5; the DYT1 signal is low and non-specific in the entire anther. (J) A spl mutant anther at late stage 5. The DYT1 signal is at the background level. (K) An ems1 mutant anther locule at late stage 5. Uniformly weak DYT1 signal can be detected in meiocytes and little signal in cells surrounding the meiocytes. (G) The sense control with a Ler late stage 5 anther. Only background signal is seen. Rt, root; Sm, stem; Lf, leaf;Se, silique; S12, stage 12 flower; Inf, inflorescence; Ar, anther; WT-Inf,wild-type inflorescence; ems1-Inf, ems1 inflorescence; spl-Inf, spl inflorescence; T, tapetum; Ms, meiocytes; I,indeterminate cells; E-Ms, excess meiocytes. Scale bars: 20 μm in C-K.

Some tapetum marker genes, such as A6 and A9, were expressed in the dyt1 mutant at slightly reduced levels. It is possible that other genes are also important for the activation of some tapetum genes. In addition to DYT1, previous reports described mutants with similar phenotypes,such as fat tapetum, gne1 and gne4(Sanders et al., 1999; Sorensen et al., 2002). Although the molecular nature of the FAT TAPETUM, GNE1 and GNE4 genes are unknown at this time, it is likely that additional loci are involved in defining the tapetal cell fate. In other words, DYT1 is essential, but not sufficient, for the specification of the tapetum identity, as supported by our observation that overexpression of DYT1 did not alter anther phenotypes in wild-type or mutant backgrounds. Recently, it was reported that the AtMYB33 and AtMYB65 genes redundantly facilitate tapetum development, with the double mutant having tapetum defects before the completion of meiosis(Millar and Gubler, 2005). It is known that bHLH transcription factors can form homodimers or heterodimers with other bHLH proteins. In some cases, it has been shown that bHLH proteins can form complexes with MYB proteins and WD-40 proteins(Ramsay and Glover, 2005). It is possible that the AtMYB33 and AtMYB65 proteins may form heterodimers with DYT1 to regulate tapetum-preferential gene expression. This idea is consistent with our result that the expression of these two MYB genes is not altered in the dyt1 mutant.

Fig. 6.

Expression of anther and tapetum genes in the wild type and dyt1 mutant. (A) Each of the 11 genes, including the controls UBQ1 and MYB4, show either normal or increased expression in the dyt1 mutant background compared with the wild type.(B,C) Twenty-one genes show significantly decreased expression in the dyt1 mutant background. (D) Real-time PCR of selected anther genes in both wild-type and dyt1 backgrounds. (E)Effects of overexpression of DYT1 on selected anther genes. No significant effects were observed by DYT1 overexpression, even though the DYT1 expression levels were elevated. (F) Expression pattern of selected putative regulatory genes in spl, ems1 and dyt1 background.

Fig. 6.

Expression of anther and tapetum genes in the wild type and dyt1 mutant. (A) Each of the 11 genes, including the controls UBQ1 and MYB4, show either normal or increased expression in the dyt1 mutant background compared with the wild type.(B,C) Twenty-one genes show significantly decreased expression in the dyt1 mutant background. (D) Real-time PCR of selected anther genes in both wild-type and dyt1 backgrounds. (E)Effects of overexpression of DYT1 on selected anther genes. No significant effects were observed by DYT1 overexpression, even though the DYT1 expression levels were elevated. (F) Expression pattern of selected putative regulatory genes in spl, ems1 and dyt1 background.

Our analysis of gene expression in the spl mutant suggest that two other genes (At2g31210 and At1g06170) encoding bHLH proteins might also act downstream of SPL/NZZ and that these bHLH genes might in turn regulate other genes. Indeed, an examination of upstream sequences (500 bp from the ATG codon) of 163 putative tapetum-preferential genes (identified from microarray data, W.Z., A. Wijeratne, Y.S. and H.M., unpublished) revealed that 143 of them have potential binding sites for bHLH proteins, and 69 genes have putative MYB-binding sites, with 59 genes have both types of elements. These observations support the hypothesis that bHLH and MYB proteins regulate overlapping but non-identical sets of tapetum-preferential genes. In particular, 18 out of 21 genes that exhibit reduced expression in the dyt1 mutant have putative bHLH-binding sites; in addition, between 500-1000 bp upstream of the AMS ATG codon, there is a bHLH-binding consensus site. Further experiments are needed to test whether these genes are direct targets of DYT1 and/or other bHLH proteins.

### DYT1 supports completion of meiosis

It is known that a functional tapetum is required for normal pollen development following meiosis, as shown by molecular ablation studies and the characterization of mutants such as ams(Aarts et al., 1997; Ito and Shinozaki, 2002; Sorensen et al., 2003; Wilson et al., 2001). In the ems1/exs, serk1 serk2 and tpd1 mutants, the tapetum is missing and excess meiocytes occupy the position of the tapetum(Albrecht et al., 2005; Colcombet et al., 2005; Yang et al., 2003; Zhao et al., 2002). Nevertheless, meiotic nuclear divisions still occur in the ems1mutant, indicating that the tapetum is not required for meiotic nuclear events. However, the ems1 meiocytes do not undergo cytokinesis,suggesting that tapetum might be needed for the completion of meiosis. The dyt1 mutant phenotypes provide further support for this idea. In addition to a morphologically abnormal tapetum, the dyt1 mutant meiocytes were found to have thinner callose cell walls than normal,suggesting that normal tapetum function is needed for the formation of the callose wall. Nevertheless, DYT1 expression was detected at a low level in the meiocytes and expression of some meiotic genes was reduced;therefore, it is possible that DYT1 might also function in meiocytes.

### A model for DYT1 function and the control of tapetum identity

This and previous studies support a model for the genetic control of tapetum development and function (Fig. 8), although evidence for biochemical interactions is not yet available. SPL/NZZ is required for the formation of sporogenous cells and surrounding somatic cell layers, including the tapetum(Yang et al., 1999). Recently,Ito et al. showed that AG is a direct activator of SPL/NZZexpression (Ito et al., 2004). EMS1/EXS, TPD1 and SERK1/SERK2 are required for the formation of tapetum (Albrecht et al.,2005; Colcombet et al.,2005; Yang et al.,2003; Zhao et al.,2002). In addition, phenotypic changes caused by TPD1overexpression are dependent on EMS1/EXS(Yang et al., 2005). We show here that SPL/NZZ and EMS1/EXS positively regulate expression of DYT1. The AtMYB33 and AtMYB65 genes are expressed in the tapetum and their expression does not require the SPL/NZZ, EMS1/EXS or DYT1 gene.

Fig. 7.

The ems1 dyt1 double mutant anther morphology at late stage 5. (A) Wild-type. (B) dyt1.(C) ems1. (D) ems1 dyt1 double mutant. The double mutant lacks the tapetum. T, tapetum; ML, middle layer; Ms, meiocytes; E-Ms, excess meiocytes; I, indeterminate cells. Scale bar: 10 μm.

Fig. 7.

The ems1 dyt1 double mutant anther morphology at late stage 5. (A) Wild-type. (B) dyt1.(C) ems1. (D) ems1 dyt1 double mutant. The double mutant lacks the tapetum. T, tapetum; ML, middle layer; Ms, meiocytes; E-Ms, excess meiocytes; I, indeterminate cells. Scale bar: 10 μm.

Fig. 8.

A model for DYT1 function and tapetum specification. The thin arrows indicate a positive genetic regulation. The arrowheads represent gene functions controlling a developmental stage. The open arrows indicate development from one stage to the next.

Fig. 8.

A model for DYT1 function and tapetum specification. The thin arrows indicate a positive genetic regulation. The arrowheads represent gene functions controlling a developmental stage. The open arrows indicate development from one stage to the next.

In addition, it is likely that the genes that depend on DYT1 for normal expression support the tapetum function that produces the enzymatic activities and materials needed for pollen development(Dickinson and Bell, 1976; Hernould et al., 1998; Liu and Dickinson, 1989; Rubinelli et al., 1998; Scott et al., 2004; Taylor et al., 1998; Zheng et al., 2003). Among the genes regulated by DYT1 are AMS and MS1, which also encode transcription factors that probably regulate late tapetum genes. Analysis of DYT1 overexpression transgenic plants and expression studies in various mutants suggest strongly that other genes are needed for normal tapetum development. Phenotypic similarities suggest that AtMYB33 and AtMYB65, as well as potentially others (such as GNE1)(Sorensen et al., 2002), may act at approximately the same step as DYT1. Therefore, DYT1is a crucial component at a key step in the regulatory network responsible for tapetum development and function (Fig. 8).

We thank J.-P. Vielle Calzada for identifying the Ds line carrying the dyt1 mutation; W. Yang, D. Zhao and D. Ye for spl, ems1 and tpd1 mutants; A. Omeis and J. Wang for plant care;D. Grove and A. Price for DNA sequencing and real-time PCR; and G. Ning, R. Haldeman and M. Hazen for assistance in preparing anther sections and for use of the Olympus BX-51 microscope and camera. We thank A. Wijeratne for help in real-time PCR; and B. Feng, C. H. Hord, W. Hu, A. Surcel, G. Wang, A. Wijeratne and L. Zahn for helpful comments on the manuscript. This work was supported by a grant from the US Department of Energy (DE-FG02-02ER15332) to H.M. and used plant materials generated with support from a National Science Foundation grant (0215923). H.M. gratefully acknowledges the support of the John Simon Guggenheim Memorial Foundation.

Aarts, M. G., Hodge, R., Kalantidis, K., Florack, D., Wilson, Z. A., Mulligan, B. J., Stiekema, W. J., Scott, R. and Pereira, A.(
1997
). The Arabidopsis MALE STERILITY 2 protein shares similarity with reductases in elongation/condensation complexes.
Plant J.
12
,
615
-623.
Albrecht, C., Russinova, E., Hecht, V., Baaijens, E. and de Vries, S. (
2005
). The Arabidopsis thaliana SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASES1 and 2 control male sporogenesis.
Plant Cell
17
,
3337
-3349.
Alexander, M. P. (
1969
). Differential staining of aborted and nonaborted pollen.
Stain Technol.
44
,
117
-122.
Azumi, Y., Liu, D., Zhao, D., Li, W., Wang, G., Hu, Y. and Ma,H. (
2002
). Homolog interaction during meiotic prophase I in Arabidopsis requires the SOLO DANCERS gene encoding a novel cyclin-like protein.
EMBO J.
21
,
3081
-3095.
Canales, C., Bhatt, A. M., Scott, R. and Dickinson, H.(
2002
). EXS, a putative LRR receptor kinase, regulates male germline cell number and tapetal identity and promotes seed development in Arabidopsis.
Curr. Biol.
12
,
1718
-1727.
Chen, C., Zhang, W., Timofejeva, L., Gerardin, Y. and Ma, H.(
2005
). The Arabidopsis ROCK-N-ROLLERS gene encodes a homolog of the yeast ATP-dependent DNA helicase MER3 and is required for normal meiotic crossover formation.
Plant J.
43
,
321
-334.
Coen, E. S. and Meyerowitz, E. M. (
1991
). The war of the whorls: genetic interactions controlling flower development.
Nature
353
,
31
-37.
Colcombet, J., Boisson-Dernier, A., Ros-Palau, R., Vera, C. E. and Schroeder, J. I. (
2005
). Arabidopsis SOMATIC EMBRYOGENESIS RECEPTOR KINASES1 and 2 are essential for tapetum development and microspore maturation.
Plant Cell
17
,
3350
-3361.
Dickinson, H. G. and Bell, P. R. (
1976
). The changes in the tapetum of Pinus banksiana accompanying formation and maturation of the pollen.
Ann. Bot.
40
,
1101
-1109.
Glover, J., Grelon, M., Craig, S., Chaudhury, A. and Dennis,E. (
1998
). Cloning and characterization of MS5 from Arabidopsis: a gene critical in male meiosis.
Plant J.
15
,
345
-356.
Goldberg, R. B., Beals, T. P. and Sanders, P. M.(
1993
). Anther development: basic principles and practical applications.
Plant Cell
5
,
1217
-1229.
Heim, M. A., Jakoby, M., Werber, M., Martin, C., Weisshaar, B. and Bailey, P. C. (
2003
). The basic helix-loop-helix transcription factor family in plants: a genome-wide study of protein structure and functional diversity.
Mol. Biol. Evol.
20
,
735
-747.
Hernould, M., Suharsono Zabaleta, E., Carde, J. P., Litvak, S.,Araya, A. and Mouras, A. (
1998
). Impairment of tapetum and mitochondria in engineered male-sterile tobacco plants.
Plant Mol. Biol.
36
,
499
-508.
Higginson, T., Li, S. F. and Parish, R. W.(
2003
). AtMYB103 regulates tapetum and trichome development in Arabidopsis thaliana.
Plant J.
35
,
177
-192.
Ito, T. and Shinozaki, K. (
2002
). The MALE STERILITY1 gene of Arabidopsis, encoding a nuclear protein with a PHD-finger motif, is expressed in tapetal cells and is required for pollen maturation.
Plant Cell Physiol.
43
,
1285
-1292.
Ito, T., Wellmer, F., Yu, H., Das, P., Ito, N., Alves-Ferreira,M., Riechmann, J. L. and Meyerowitz, E. M. (
2004
). The homeotic protein AGAMOUS controls microsporogenesis by regulation of SPOROCYTELESS.
Nature
430
,
356
-360.
Jung, K. H., Han, M. J., Lee, Y. S., Kim, Y. W., Hwang, I., Kim,M. J., Kim, Y. K., Nahm, B. H. and An, G. (
2005
). Rice UNDEVELOPED TAPETUM1 is a major regulator of early tapetum development.
Plant Cell
17
,
2705
-2722.
Kumar, S., Tamura, K. and Nei, M. (
2004
). MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment.
Brief. Bioinformaics
5
,
150
-163.
Li, J., Jia, D. and Chen, X. (
2001
). HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear RNA binding protein.
Plant Cell
13
,
2269
-2281.
Li, W., Chen, C., Markmann-Mulisch, U., Timofejeva, L.,Schmelzer, E., Ma, H. and Reiss, B. (
2004
). The Arabidopsis AtRAD51 gene is dispensable for vegetative development but required for meiosis.
101
,
10596
-10601.
Liu, X. C. and Dickinson, H. G. (
1989
). Cellular energy levels and their effect on male cell abortion in cytoplasmically male sterile lines of Petunia hybrida.
Sex Plant Reprod.
2
,
167
-172.
Liu, Y. G. and Whittier, R. F. (
1995
). Thermal asymmetric interlaced PCR: automatable amplification and sequencing of insert end fragments from P1 and YAC clones for chromosome walking.
Genomics
25
,
674
-681.
Ma, H. (
2005
). Molecular genetic analyses of microsporogenesis and microgametogenesis in flowering plants.
Annu. Rev. Plant Biol.
56
,
393
-434.
Mercier, R., Jolivet, S., Vezon, D., Huppe, E., Chelysheva, L.,Giovanni, M., Nogue, F., Doutriaux, M. P., Horlow, C., Grelon, M. et al.(
2005
). Two meiotic crossover classes cohabit in Arabidopsis: one is dependent on MER3,whereas the other one is not.
Curr. Biol.
15
,
692
-701.
Millar, A. A. and Gubler, F. (
2005
). The Arabidopsis GAMYB-like genes, MYB33 and MYB65, are microRNA-regulated genes that redundantly facilitate anther development.
Plant Cell
17
,
705
-721.
Ni, W., Xie, D., Hobbie, L., Feng, B., Zhao, D., Akkara, J. and Ma, H. (
2004
). Regulation of flower development in Arabidopsis by SCF complexes.
Plant Physiol.
134
,
1574
-1585.
Preston, J., Wheeler, J., Heazlewood, J., Li, S. F. and Parish,R. W. (
2004
). AtMYB32 is required for normal pollen development in Arabidopsis thaliana.
Plant J.
40
,
979
-995.
Ramsay, N. A. and Glover, B. J. (
2005
). MYB-bHLH-WD40 protein complex and the evolution of cellular diversity.
Trends Plant Sci.
10
,
63
-70.
Ross, K. J., Fransz, P. and Jones, G. H.(
1996
). A light microscopic atlas of meiosis in Arabidopsis thaliana.
Chromosome Res.
4
,
507
-516.
Rubinelli, P., Hu, Y. and Ma, H. (
1998
). Identification, sequence analysis and expression studies of novel anther-specific genes of Arabidopsis thaliana.
Plant Mol. Biol.
37
,
607
-619.
Sanders, P. M., Bui, A. Q., Weterings, K., McIntire, K. N., Hsu,Y. C., Lee, P. Y., Truong, M. T., Beals, T. P. and Goldberg, R. B.(
1999
). Anther developmental defects in Arabidopsis thaliana male-sterile mutants.
Sex Plant Reprod.
11
,
297
-322.
Schiefthaler, U., Balasubramanian, S., Sieber, P., Chevalier,D., Wisman, E. and Schneitz, K. (
1999
). Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana.
96
,
11664
-11669.
Scott, R. J., Spielman, M. and Dickinson, H. G.(
2004
). Stamen structure and Function.
Plant Cell
16
,
S46
-S60.
Sorensen, A., Guerineau, F., Canales-Holzeis, C., Dickinson, H. G. and Scott, R. J. (
2002
). A novel extinction screen in Arabidopsis thaliana identifies mutant plants defective in early microsporangial development.
Plant J.
29
,
581
-594.
Sorensen, A. M., Krober, S., Unte, U. S., Huijser, P., Dekker,K. and Saedler, H. (
2003
). The Arabidopsis ABORTED MICROSPORES (AMS) gene encodes a MYC class transcription factor.
Plant J.
33
,
413
-423.
Taylor, P. E., Glover, J. A., Lavithis, M., Craig, S., Singh, M. B., Knox, R. B., Dennis, E. S. and Chaudhury, A. M. (
1998
). Genetic control of male fertility in Arabidopsis thaliana: structural analyses of postmeiotic developmental mutants.
Planta
205
,
492
-505.
Toledo-Ortiz, G., Huq, E. and Quail, P. H.(
2003
). The Arabidopsis basic/helix-loop-helix transcription factor family.
Plant Cell
15
,
1749
-1770.
Vannini, C., Locatelli, F., Bracale, M., Magnani, E., Marsoni,M., Osnato, M., Mattana, M., Baldoni, E. and Coraggio, I.(
2004
). Overexpression of the rice OsMYB4 gene increases chilling and freezing tolerance of Arabidopsis thaliana plants.
Plant J.
37
,
115
-127.
Wilson, Z. A., Morroll, S. M., Dawson, J., Swarup, R. and Tighe,P. J. (
2001
). The Arabidopsis MALE STERILITY1 (MS1)gene is a transcriptional regulator of male gametogenesis, with homology to the PHD-finger family of transcription factors.
Plant J.
28
,
27
-39.
Yang, S. L., Xie, L. F., Mao, H. Z., Puah, C. S., Yang, W. C.,Jiang, L., Sundaresan, V. and Ye, D. (
2003
). TAPETUM DETERMINANT1 is required for cell specialization in the Arabidopsis anther.
Plant Cell
15
,
2792
-2804.
Yang, S. L., Jiang, L., Puah, C. S., Xie, L. F., Zhang, X. Q.,Chen, L. Q., Yang, W. C. and Ye, D. (
2005
). Overexpression of TAPETUM DETERMINANT1 alters the cell fates in the Arabidopsis carpel and tapetum via genetic interaction with EXCESS MICROSPOROCYTES1/EXTRA SPOROGENOUS CELLS.
Plant Physiol.
139
,
186
-191.
Yang, W. C., Ye, D., Xu, J. and Sundaresan, V.(
1999
). The SPOROCYTELESS gene of Arabidopsisis required for initiation of sporogenesis and encodes a novel nuclear protein.
Genes Dev.
13
,
2108
-2117.
Zhao, D. Z., Wang, G. F., Speal, B. and Ma, H.(
2002
). The EXCESS MICROSPOROCYTES1 gene encodes a putative leucine-rich repeat receptor protein kinase that controls somatic and reproductive cell fates in the Arabidopsis anther.
Genes Dev.
16
,
2021
-2031.
Zheng, Z., Xia, Q., Dauk, M., Shen, W., Selvaraj, G. and Zou,J. (
2003
). Arabidopsis AtGPAT1, a member of the membrane-bound glycerol-3-phosphate acyltransferase gene family, is essential for tapetum differentiation and male fertility.
Plant Cell
15
,
1872
-1887.